The present invention concerns an animal model for human and non-human diseases caused by Apicomplexan parasites including but not limited to Sarcocystis neurona, Sarcocystis dasypus (syn S. neurona), Sarcocystis cruzi, Sarcocystis falcatula, Sarcocystis sp., Toxoplasma gondii, Neospora caninum, N. hughesi, Eimeria and Plasmodium.
The following is a list of prior art references considered to be pertinent for the subsequent description:
1. Dubey J. P., Davis S. W., Speer C. A., Bowman D. D., de Lahunta A., Granstrom D. E., Topper M. J., Hamir A. N., Cummings J. F., and Suter M. M. 1991. Sarcocystis Neurona N. SP. (Protozoa: Apicomplexa), The etiologic Agent of Equine Protozoal myeloencephalitis. J. Parasitol., 77(2): 212-218.
2. Blythe, L. L., Granstrom, D. E., Hansen, D. E., Walker, L. L., Bartlett, J., and Stamper, S. 1997. Seroprevalence of antibodies to Sarcocystis neurona in horses residing in Oregon. JAVMA, 210(4): 525-528.
3. Bentz, B. G., Granstrom, D. E., Stamper, S. 1997. Seroprevalence of antibodies to Sarcocystis neurona in horses residing in a county of southeastern Pennsylvania. JAVMA, 210(4): 517-518.
4. Saville, W. J., Reed , S. M., Granstrom, D. E., Hinchcliff, K. W., Kohn, C. W., Wittum, T. E., and Stamper, S. 1997. Seroprevalence of antibodies to Sarcocystis neurona in horses residing in Ohio. JAVMA, 210(4): 519-523.
5. Gray L. C., Magdesian, K. G., Sturges, B. K., Madigan, J. E. 2001. Suspected protozoal myeloencephalitis in a two-month-old colt. Vet Rec. 2001 September 1; 149(9):269-73.
6. MacKay, R. J. 1997. Serum antibodies to Sarcocystis neuronaxe2x80x94half the horses in the United States have them! JAVMA, 210(4): 482-483.
7. Dubey, A. P. 1976. A review of Sarcocystis of Domestic Animals. JAVMA, 169 (10):1061-1078.
8. Dubey, J. P., 1986. Equine protozoal myeloencephalitis in a pony. JAVMA, 188:1311-1312.
9. Granstrom, D. E, Saville, W. J. 1998. Equine Protozoal Myeloencephalitis In: S. M. Reed and W. M. Bailey, eds. Equine Internal Medicine. Philadelphia, Pa.: WB Saunders Company, 486-491.
10. Fenger, C. K., Granstrom, D. E., Gajadhar, A. A., Williams, N. M., McCrillis, S. A., Stamper, S., Langemeier, J. L., Dubey, J. P. 1997. Experimental induction of equine protozoal myeloencephalitis in horses using Sarcocystis sp. sporocysts from the opossum (Didelphis virginiana). Vet Parasitol., 68:199-213.
11. O""Donoghue, P., Lumb, R., Smith, P., Brooker, J., Mencke, N. 1990. Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy. Vet. Immunol. Immunopathol., 24(1):11-25.
12. Marsh, A. E., Barr, B. C., Tell, L., Koski, M., Greiner, E., Dame, J. and Conrad, P. A. 1997. In vitro cultivation and experimental inoculation of Sarcocystis falcatula and Sarcocystis neurona merozoites into budgerigars (Melopsittacus undulatus). J. Parasitol., 83(6): 1189-1192.
13. Dubey J R, Rosypal A C, Rosenthal B M, Thomas N J, Lindsay D S, Stanek J F, Reed S M Saville W J. 2001. Sarcocystis neurona infections in sea otter (Enhydra lutris): evidence for natural infections with sarcocysts and transmission of infection to opossums (Didelphis virginiana). J Parasitol December; 87(6):1387-93.
14. Rosypal A C, Lindsay D S, Duncan R, Ansar Ahmed S, Zajac A M, Dubey J P. 2002 Mice lacking the gene for inducible or endothelial nitric oxide are resistant to sporocyst induced Sarcocystis neurona infections. Vet Parasitol February 4;103(4):315-21.
15. Dubey J R, Rosypal A C, Rosenthal B M, Thomas N J, Lindsay D S, Stanek J F, Reed S M, Saville W J. 2001. Sarcocystis neurona infections in sea otter (Enhydra lutris): evidence for natural infections with sarcocysts and transmission of infection to opossums (Didelphis virginiana). J Parasitol December; 87(6):1387-93
16. Rosypal A C, Lindsay D S, Duncan R, Ansar Ahmed S, Zajac A M, Dubey J P. 2002 Mice lacking the gene for inducible or endothelial nitric oxide are resistant to sporocyst induced Sarcocystis neurona infections. Vet Parasitol February 4;103(4):315-21
17. Cheadle M A, Ginn P E, Lindsay D S, Greiner E C. 2002. Neurologic disease in gamma-interferon gene knockout mice caused by Sarcocystis neurona sporocysts collected from opossums fed armadillo muscle. Vet Parasitol January 3;103(1-2):65-9
18. Dubey J P, Lindsay D S, Kwok O C, Shen S K. 2001. The gamma interferon knockout mouse model for sarcocystis neurona: comparison of infectivity of sporocysts and merozoites and routes of inoculation. J Parasitol October; 87(5):1171-3
19. Lindsay D S, Dubey J P. 2001. Determination of the activity of pyrantel tartrate against Sarcocystis neurona in gamma-interferon gene knockout mice. Vet Parasitol May 22;97(2):141-4
20. Dubey J P. 2001. Migration and development of Sarcocystis neurona in tissues of interferon gamma knockout mice fed sporocysts from a naturally infected opossum. Vet Parasitol February 26;95(2-4):341-51
21. Speer C A, Dubey J P. 2001. Ultrastructure of schizonts and merozoites of Sarcocystis neurona. Vet Parasitol February 26;95(2-4):263-71
22. Cheadle M A, Tanhauser S M, Scase T J, Dame J B, Mackay R J, Ginn P E, Greiner E C. 2001. Viability of Sarcocystis neurona sporocysts and dose titration in gamma-interferon knockout mice. Vet Parasitol February 26;95(2-4):223-31
23. Dubey J P, Mattson D E, Speer C A, Hamir A N, Lindsay D S, Rosenthal B M, Kwok O C, Baker R J, Mulrooney D M, Tornquist S J, Gerros T C. 2001. Characteristics of a recent isolate of Sarcocystis neurona (SN7) from a horse and loss of pathogenicity of isolates SN6 and SN7 by passages in cell culture. Vet Parasitol February 26;95(2-4):155-66
24. Tenter , A. M., Johnson, M. R., Zimmerman, G. L. 1989. Differentiation of Sarcocyst species in European sheep by isoelectric focusing. Parasitol. Res. 76(2):107-114.
25. Sommer, I., Horn, K., Heydorn, A. O., Mehlhorn, H., Ruger, W. 1992. Acomparison of sporozoite and cyst merozoite surface proteins of Sarcocystis. Parasitol Res., 78:398-403.
26. Dubey, J. P., Kistner, T. P., and Callis, G. 1983. Development of Sarcocystis in mule deer transmitted through dogs and coyotes. Can. J. Zool., 61:2904-2912.
27. O""Donoghue, P. J. and Ford, G. E. 1984. The asexual pre-cyst development of Sarcocystis tenella in experimentally infected specific-pathogen-free lambs. Int. J. Parasitol., 14(4):345-355.
28. Speer, C. A. and Dubey, J. P. 1981. An ultrastructural study of first and second generation merogony in the coccidian Sarcocystis tenella. J. Protozool., 28(4):424-431.
29. Johnson, A. J., Hildebrandt, P. K., and Fayer, R. 1975. Experimentally induced Sarcocystis infection in calves. Pathology Am. J. Vet. Res., 36(7):995-999.
30. Fayer, R. and Leek, R. G. 1979. Sarcocystis transmitted by blood transfusion. J Parasitol., 65(6):890-893.
31. Ellison, S. P., Omara-Opyeme, A. L., Yowell, Yowell, C. A., Marsh, A. E., Dame, J. B. 2002. Molecular characterization of a major 29 kDa surface antigen of Sarcocystis neurona. Int. J. Parasit. 32: 217-225.
32. Dubey, J. P., and Lindsay D. S. 1998. Isolation in immunodeficient mice of Sarcocystis neurona from opossum (Didelphis viriniana) feces and its differentiation from Sarcocystis falcatula. International Journal for Parasitology. 28:1823-8.
33. NAHMS. 2001. Equine Protozoal Myeloencephalitis (EPM) in the U.S. USDA:APHIS:VS, CEAH, National Animal Health Monitoring System. Fort Collins, Colo. #N312.0501.
34. Lindsay D S, Dykstra C C, Williams A, Spencer J A, Lenz S D, Palma K, Dubey J P, Blagburn B L. 2000 Inoculation of Sarcocystis neurona merozoites into the central nervous system of horses. Vet Parasitol. September 20;92(2): 157-63.
35. Ellison, S. P., Greiner, E., Dame, J. B. 2000. In vitro culture and synchronous release of Sarcocystis neurona merozoites from host cells. Vet. Parasitol. 1982: 1-11.
36. Speer, C. A., Dubey, J. P. 2001. Ultrastructure of schizonts and merozoites of Sarcocystis neurona. Vet Parasitol. 95: 263-271.
37. Dubey, J. P., Mattson, D. E., Speer, C. A., Hamir, A. N., Lindsay, D. S., Rosenthal, B. M., Kwok, O. C., Baker, R. J., Mulrooney, D. M., Tornquist, S. J., Gerros, T. C. 2001. Characteristics of a recent isolate of Sarcocystis neurona (SN7) from a horse and loss of pathogenicity of isolates SN6 and SN7 by passages in cell culture. Vet Parasitol. February 26;95(2-4):155-66
The acknowledgement herein of any of the above references is to allow the reader to gain appreciation of the prior art. The acknowledgement should, however, not be construed as an indication that these references are in any way relevant to the issue of patentability of the invention as defined in the appended claims.
Acknowledgement of the above references will be made by indicating the number from the above list.
The development of effective treatments, therapies or diagnostics for Apicomplexan parasite diseases has been hampered by lack of suitable models for reproduction of the disease. This is at least in part due to the fact the Apicomplexan parasites such as Sarcocystis neurona cause diseases in immunologically privileged compartments such as the brain, spinal cord or fetal tissues, where the mammalian body cannot easily stimulate an immune response to control parasite invasion of these compartments. Illustratively, Equine Protozoal Myeloencephalitis (EPM) which is the leading infectious neurologic or abortigenic equine disease in the Western Hemisphere is caused by the Apicomplexan parasite Sarcocystis neurona (S. neurona). While the symptoms and effects of EPM have been recognized since the 1970""s, it was not until 1991 that the protozoan parasite that causes EPM was isolated and cultured from a horse and given the name Sarcocystis neurona[1]. Sarcocystis neurona (S. neurona), recently recognized as S. dasypus (syn. S. neurona) cycles naturally between opossums and armadillos/raccoons. Recent investigations indicate that the feces of the opossum (the definitive host) may be the source of the infection for horses. Thus, the horse is an aberrant host, becoming exposed when it consumes infectious material from opossum feces. An aberrant host is a dead-end host, as infectious forms of the parasite are not passed from horse to horse or from infected horse to a definitive or true intermediate host. Incidence of EPM is likely greatest in areas with high opossum populations. EPM appears to have a sporadic distribution, although outbreaks have been reported on farms in Kentucky, Ohio, Indiana, Michigan and Florida [2-4].
A horse of any age, breed, or sex may be affected by EPM. The disease has been reported in a horse as young as two months of age, as well as one in its thirties [5]. In fact, any horse demonstrating neurologic abnormalities may be infected with an EPM-producing organism. In the horse, the most prominent EPM-producing organism, S. neurona, does not produce clinical signs of disease as a result of cyst formation, but as the cysts (sporozoites) convert to merozoites which make their way to the brain and spinal cord, where they proliferate and cause clinical disease. Clinical signs of a horse with EPM do not develop until the organism has crossed the blood brain barrier and is within the central nervous system. These signs include weakness, muscle atrophy, spinal ataxia, or xe2x80x9cwobblingxe2x80x9d and/or head tilt with asymmetry of the face (e.g., eyelid, ear, or lip). A severely EPM-affected horse may go down and be unable to rise. Lameness not traceable to orthopedic disease or any combination of the above signs may occur in early or less severe infections. In most cases, an affected horse is bright and alert with a normal appetite, hematological and biochemical blood values are usually in the normal range.
Surveys (using a positive serum test to immunoblotted S. neurona antigens to indicate exposure to the parasite) which were conducted in central Kentucky, one county in Pennsylvania and the entire states of Ohio and Oregon, have revealed that approximately fifty percent (50%) of the horses in the surveyed areas have been exposed to S. neurona[2-4,6]. However, a positive test result on the immunoblot test does not necessarily indicate the presence of an active form of the disease. The incidence of the active disease appears to be much lower than the seroprevalence since less than 1% of seropositive horses are clinically affected [6]. At any rate, epidemiology and economic significance of S. neurona infection is substantial. Of animals clinically affected, 30-40% reportedly fail to respond to current therapy, and some of these animals die [6]. Conventional therapy relies on relatively non-specific drug/medications and/or combinations, the efficacy of which cannot be optimized because of the lack of a model of the disease. As such better and more effective prophylactic, or therapeutic modalities are required but cannot be thoroughly tested without an animal model that can predictably reproduce disease.
Currently, the only methods useful for diagnosis of this disease require removing cerebraispinal fluid from the horsexe2x80x94a method that is highly invasive and carries some danger. Originally, the diagnosis was based on the presence of antibodies to S. neurona in serum, though it is now known that a positive serum test cannot be used to make a diagnosis. Such positive serum test simply indicates exposure to the parasite, not necessarily presence of the disease. Cerebral spinal fluid (CSF) testing is now believed to be the most useful test to assist in the diagnosis of this disease in a live horse [10]. However, even this test is flawed as clinically normal and vaccinated horses still seem to contain antibodies in the CSF. An improved animal model will be useful to develop less invasive diagnostic assays for EPM as well as to elucidate the immune response to vaccination and determine if the vaccine is efficacious
In the case of EPM, sporocyst challenged horses are reported to produce antibodies in the CSF and this was taken as evidence of the parasites entry into the CNS [11]. However, neither isolation of parasites nor the presence of parasites in the CNS or any other tissue from the challenged horses was demonstrated. Therefore, Koch""s postulate was not demonstrated, the disease EPM was not produced and the hoped-for model was not acceptable for the purpose of making drugs, vaccines and diagnostics.
An animal model, which has been used to date to study S. neurona isolated from natural cases of EPM, includes infection of nude mice or interferon gamma knock-out mice with sporocysts or culture derived merozoites [12]. These experiments result in neurological signs and isolation of the organism from the CNS [13-25]. However, the relevancy of this model is doubtful since these mice are immuno-deficient; thus any immuno-based selection forces acting in normal animals are absent.
From the foregoing, it would be realized that despite a great deal of past and on-going effort, there remains an unfulfilled need for an animal model for Apicomplexan parasitic diseases including EPM.
In accordance with the foregoing, the present invention encompasses an animal model comprising reproducing clinical signs of Apicomplexan parasite disease by providing an Apicomplexan parasite which is incorporated in a host cell, such as a host lymphocyte cell, in an amount that is effective to cross the brain or placental barrier of the host, when the host is inoculated with the parasite-incorporated cell. The present invention is directed to a unique discovery that mammalian cells, specifically lymphocytes of all types and any other cell that can be activated so as to cross the blood brain barrier or the placental barrier, can be infected by merozoites of Apicomplexan organisms. After merozite infection of the mammalian cells, the infected cells can be used to infect mammals, reproduce clinical signs of Apicomplexan diseases and serve as models that are required for development of efficacious drug treatments, prophylactic modalities such as drugs and vaccines, and diagnostic tests for determination of Apicomplexan infections. Apicomplexan diseases described by the present invention include but are not limited to Sarcocystis, Toxoplasma and Neospora.
More specifically, the present invention is directed to an animal model for S. neurona that produces infection in the CNS of horses and reproduces the clinical signs of EPM. Without being bound to any particular theory of the invention, this model is based on the discovery that virulent merozoite cells of S. neurona can be induced to enter certain mammalian cells cultured in vitro and, as long as they retain their unextruded conoid, they can be transferred back to the homologous host. After transfer, the homologous host cells infected with virulent merozoite cells migrate via the blood to the central nervous system and can cross the blood brain barrier. The clinical signs of disease observed are produced by the virulent merozoites. In addition to the clinical signs, an animal model must produce serum and CSF antibodies against the EPM-producing organism, provide for isolation of the organism from the spinal tissues and/or CSF fluid in in vitro culture, and demonstration of the organism in the tissues of the horse.
Additionally, the present invention is directed to an animal model for S. neurona that produces infection in the fetus of pregnant mammals, potentially causing death of the fetus and/or abortion. This model is based on the discovery that virulent merozoite cells of S. neurona can be induced to enter certain mammalian cells cultured in vitro and, as long as they retain their unextruded conoid, they can be transferred back to the homologous pregnant host. After transfer to the homologous host, the homologous host cells infected with virulent merozoite cells migrate via the blood to the placenta and can cross the placental barrier. The clinical signs of disease observed are produced by the virulent merozoites infecting the fetus. For this model it is preferable to infect the filly or mare during the first trimester of gestation.
It is a distinct feature of the present invention that specific mammalian cells are infected in vitro with an EPM-producing parasite, allowed to reach the stage of growth in which the merozoite stage of the parasite is optimally virulent, and is used to cross the blood brain barrier. It is also a distinct feature of the invention that it has now been recognized that the Apicomplexan parasites can evade the immune response and to produce clinical signs of disease.
The present invention provides animal models for Apicomplexan infections including but not limited to Toxoplasma, Neospora, and/or Sarcocystis.
The present invention provides the models for development of preventive and/or therapeutic agents (drugs and vaccines) by demonstrating their effectiveness against Apicomplexan infections such as Toxoplasma, Neospora, and/or Sarcocystis.
The present invention also provides a method for developing diagnostic tests and determining their effectiveness in Apicomplexan infections such as Toxoplasma, Neospora, and/or Sarcocystis.
The present invention provides the animal model for the development of a new diagnostic test to diagnose EPM in the horse by measuring the xcex3-interferon response of horses to an antigen from an EPM-producing organism.
The present invention provides the animal model for the development of a new diagnostic test to diagnose EPM in the horse by measuring the antibody response to SAG-1, the outer membrane protein of S. neurona, in the CSF and/or serum.
1. xe2x80x9cIsolationxe2x80x9d as used herein means recovery and in vitro growth of an organism from a clinically-diseased animal.
2. xe2x80x9cBlood brain barrierxe2x80x9d as used herein means the anatomical barrier to all molecules composed of two membranes in series: the lumenal and the ablumenal membranes of the brain capillary endothelial cell, which are separated by approximately 300 nm of endothelial cytoplasm. The blood brain barrier also consists of transport systems on both lumenal and ablumenal membranes of the endothelial cell for solute transcytosis from blood to brain. The blood brain barrier also contains a number of specialized carrier transport systems within the blood brain barrier that mediate brain uptake of circulating nutrients, such as glucose, amino acids, choline, transport or diapedesis across the anatomical barrier is allowed.
3. xe2x80x9cPlacental barrierxe2x80x9d as used herein means the anatomical barrier to all molecules and components of two membranes in series that protects the fetus from infection and/or toxic substances.
4. xe2x80x9cVirulent merozoite cellxe2x80x9d as used herein means a merozoite producing proteins for attachment and invasion of host cells so that the parasite can attach to and enter a host cell where it replicates and responds to cell signals (parasite and host) resulting in the release of prodgeny capable of infecting more host cells.
5. xe2x80x9cConoid formxe2x80x9d as used herein means the physical state of the apicomplex of parasites consisting of the conoid, conoid proteins, and actin/myosin fibers that attach to a host cell and produce infection wherein the conoid is maintained in an unextruded state.
6. xe2x80x9cParasite activated lymphocytesxe2x80x9d as used herein means host cell lymphocytes that are capable of recognizing parasites, parasite antigens, or parasite proteins that are secreted. and by recognition, are capable of attaching to, engulphing, or responding to these components to result in infection of the host lymphocyte or modulation of the host immune system. 6. Homologous host as used herein means each animal from which a newly isolated cell or cell line has been obtained.
7. xe2x80x9cOptimally virulentxe2x80x9d as used herein means that the Apicomplexan merozoite is maintained in the conoid form.
8. xe2x80x9cModulate the immune systemxe2x80x9d as used herein means to orchestrate the immune system and its cascade of cellular, intercellular and intracellular reactions in a manner that favors the replication of the parasite.